Abstract: 【Objective】This study aimed to clarify the effects of Tobacco mosaic virus (TMV) content and disease grade on the disease development dynamics and defense enzyme activities in tobacco, so as to provide a theoretical basis for the early warning and management of tobacco mosaic disease.【Method】Using flue-cured tobacco variety Yunyan 87 as the test material, experiments were conducted combining artificial indoor TMV inoculation and natural field TMV infection.Six TMV concentration gradients were set in the indoor inoculation trial: 1.67, 0.835, 0.418, 0.209, 0.104, and 0.052 μg/mL, to analyze the dynamic relationship between TMV content and disease grade in tobacco plants.Field investigations were carried out in Nanyang and Pingdingshan using the five-point sampling method. Combined with indoor inoculation results, the effects of tobacco disease grade on leaf TMV content, activities of defense enzymes including PAL, POD and CAT, and MDA content were further explored.【Result】TMV content in tobacco plants increased significantly from disease grade 1 to 9 under both artificial inoculation and natural field infection conditions.The coefficient of variation of virus content was the largest from grade 1 to grade 3, with values of 0.321, 0.237 and 0.089 under artificial inoculation, natural infection in Pingdingshan and natural infection in Nanyang, respectively.The activities of PAL, POD and CAT increased with disease grade, peaked at grade 7, and then decreased, while MDA content continued to rise with increasing disease grade.【Conclusion】This study revealed the dynamic trend between TMV content and tobacco disease grade, as well as the response characteristics of defense enzyme activities and MDA content at different disease grades, providing a theoretical basis for the classified prevention and control of tobacco mosaic disease.